For a number of years, we have been interested in the regulation of respiration at a level not only under resting conditions but also during stress. Although we and others have previously examined the effect of hypoxia on cardiorespiratory function in the newborn and older animal at the system or organ level, it is not known how brainstem neurons respond to O2 deprivation or what the mechanistic basis is for the response. Recently, we have started to examine neuronal excitability during hypoxia in a brain slice preparation and made a number of very interesting preliminary observations on the transmembrane ionic fluxes that occur in brainstem neurons. Because of interactions between synaptic input and postsynaptic membrane properties in a slice preparation, the interpretation of the hypoxia-induced alterations in the biophysical properties of brainstem neurons can, however, be complicated. Thus, we developed techniques to study freshly dissociated brainstem neurons, in total isolation from synaptic input. Using this approach, we will 1) study the inherent response of single brainstem neurons and 2) investigate the cellular and molecular mechanisms that are at the basis of the responses exhibited by adult and newborn brainstem neurons (Hypoglossal, Vagal motoneurons) and, for comparative purposes, hippocampal or cortical neurons, especially that these latter cells are considered hypoxia- sensitive. In this proposal, we consider the following major hypotheses: A) Graded hypoxia induces graded electrophysiologic responses in brainstem neurons that are different from those in the hippocampus; B) the inherent neuronal response is a result of a direct effect of O2 deprivation on intracellular metabolism and membrane function; C) O2 deprivation induces an increase in Ca++i in the adult but not in the newborn and D) adult brainstem neurons regulated pHi differently from neonatal neurons and from hippocampal CA1 during hypoxia. All electrophysiological and optical techniques (microelectrode and patch-clamp, current and voltage clamp, ion- selective methods, confocal microscopy and fluorometry) are operative and both the brain slice preparation and the acute neuronal dissociation are now done on a routine basis in our laboratories. We believe that our proposed studies will lead to a better understanding of how brainstem neurons adjust to O2 deprivation; this is important since it is the activity of these same neurons that are responsible for alleviating the condition of hypoxia. These studies will allow us also to gain fundamental biologic insight into the molecular mechanisms that render an adult nerve cell more tolerant to hypoxia than another or more vulnerable to the same stress than a neonatal one.